CRISPRi-mediated silencing of the ABC transporter Ms_5779 compromises cell envelope integrity and impairs intracellular host lipid exploitation

Background Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) poses a severe global health threat, yet the contribution of cell envelope barriers to intrinsic drug tolerance remains poorly understood. The ABC protein Rv0820 is conserved across mycobacteria but functionally uncharacterized, presenting a structural paradox: it lacks canonical transmembrane helices, making conventional efflux pump function mechanistically implausible. Methods Using CRISPR interference (CRISPRi), we silenced Ms_5779 —the Mycobacterium smegmatis (M. smegmatis) ortholog of Rv0820 —to two graded depths (81% and 69% repression) and subjected the resulting strains to systematic phenotypic interrogation spanning drug susceptibility, cell envelope permeability, biofilm formation, multi-stress tolerance, and macrophage infection. Results Ms_5779 silencing did not affect growth kinetics but markedly disrupted cell envelope integrity, as evidenced by impaired biofilm formation, dissipated membrane potential, and increased ethidium bromide permeability. Silenced strains exhibited broad-spectrum drug hypersusceptibility across six antibiotic classes, with the most striking effect being a 32-fold reduction in norfloxacin MIC, alongside heightened sensitivity to oxidative, acidic, and thermal stress. In a RAW264.7 macrophage infection model, Ms_5779 silencing attenuated intracellular survival, impaired host cholesterol exploitation and foamy macrophage induction, and reduced apoptosis induction. Conclusion Collectively, these findings strongly suggest that Ms_5779 /Rv0820 is a critical functional contributor indispensable for the maintenance of cell envelope integrity, presenting a distinct mechanism from a conventional efflux pump. Targeting this essential uncharacterized ABC protein would simultaneously compromise the drug permeability barrier and the intracellular niche, nominating Rv0820 as a mechanistically distinct therapeutic target for anti-tuberculosis drug discovery.